Fieschi F, Torrents E, Toulokhonova L, Jordan A, Hellman U, Barbe J, Gibert I, Karlsson M, Sjöberg B M
Department of Molecular Biology, Stockholm University, S-106 91 Stockholm, Sweden.
J Biol Chem. 1998 Feb 20;273(8):4329-37. doi: 10.1074/jbc.273.8.4329.
Ribonucleotide reductases (RNRs) are key enzymes in living cells that provide the precursors of DNA synthesis. The three characterized classes of RNRs differ by their metal cofactor and their stable organic radical. We have purified to near homogeneity the enzymatically active Mn-containing RNR of Corynebacterium ammoniagenes, previously claimed to represent a fourth RNR class. N-terminal and internal peptide sequence analyses clearly indicate that the C. ammoniagenes RNR is a class Ib enzyme. In parallel, we have cloned a 10-kilobase pair fragment from C. ammoniagenes genomic DNA, using primers specific for the known class Ib RNR. The cloned class Ib locus contains the nrdHIEF genes typical for class Ib RNR operon. The deduced amino acid sequences of the nrdE and nrdF genes matched the peptides from the active enzyme, demonstrating that C. ammoniagenes RNR is composed of R1E and R2F components typical of class Ib. We also show that the Mn-containing RNR has a specificity for the NrdH-redoxin and a response to allosteric effectors that are typical of class Ib RNRs. Electron paramagnetic resonance and atomic absorption analyses confirm the presence of Mn as a cofactor and show, for the first time, insignificant amounts of iron and cobalt found in the other classes of RNR. Our discovery that C. ammoniagenes RNR is a class Ib enzyme and possesses all the highly conserved amino acid side chains that are known to ligate two ferric ions in other class I RNRs evokes new, challenging questions about the control of the metal site specificity in RNR. The cloning of the entire NrdHIEF locus of C. ammoniagenes will facilitate further studies along these lines.
核糖核苷酸还原酶（RNRs）是活细胞中的关键酶，可提供DNA合成的前体。已鉴定的三类RNRs因其金属辅因子和稳定的有机自由基而有所不同。我们已将产氨棒杆菌含锰的具有酶活性的RNR纯化至接近同质，该酶先前被认为代表第四类RNR。N端和内部肽序列分析清楚地表明，产氨棒杆菌RNR是I b类酶。同时，我们使用针对已知I b类RNR的特异性引物，从产氨棒杆菌基因组DNA中克隆了一个10千碱基对的片段。克隆的I b类基因座包含I b类RNR操纵子典型的nrdHIEF基因。nrdE和nrdF基因推导的氨基酸序列与活性酶的肽段匹配，表明产氨棒杆菌RNR由I b类典型的R1E和R2F组分组成。我们还表明，含锰的RNR对NrdH-氧化还原蛋白具有特异性，并且对I b类RNR典型的变构效应物有反应。电子顺磁共振和原子吸收分析证实了锰作为辅因子的存在，并首次表明在其他类别的RNR中发现的铁和钴含量极少。我们发现产氨棒杆菌RNR是I b类酶，并且具有所有高度保守的氨基酸侧链，已知这些侧链在其他I类RNR中连接两个铁离子，这引发了关于RNR中金属位点特异性控制的新的、具有挑战性的问题。产氨棒杆菌整个NrdHIEF基因座的克隆将有助于沿着这些方向进行进一步研究。
